Cytokinesis, the physical division of one cell into two daughter cells, is the final stage of the cell reproductive cycle and the least well understood. Correctly timing the process of cytokinesis so that it occurs only after chromosome replication and segregation is necessary to prevent catastrophic genomic instability and accordingly, cytokinesis is strictly regulated in concert with other events of the cell cycle. We have made significant progress in identifying and characterizing proteins essential for cytokinesis using a leading model organism for cytokinesis studies, the fission yeast Schizosaccharomyces pombe. We now propose to gain a better understanding of how these myriad proteins are controlled. A Hippo-related protein kinase cascade termed the septation initiation network (SIN) controls assembly, maintenance, and constriction of the actomyosin contractile ring and is therefore the immediate upstream regulator of cytokinesis in S. pombe. Our knowledge is limited however as to what ultimately ensures the coordination of SIN activity and thus cytokinesis, with other events of mitosis. Among other mechanisms, SIN function is directly inhibited by the ubiquitin ligase Dma1 and this comprises a second branch of mitotic checkpoint signaling. Given our new understanding that Dma1 is a general inhibitor of the polo-like kinase, Plo1 at mitotic initiation, we will study whether this mechanism of controlling mitotic inhibition is shared with the human tumor suppressor CHFR. We will determine how Dma1 activity and localization are controlled throughout the cell cycle by upstream pathways including one comprising CK1. We propose a comprehensive set of biochemical and cell biological experiments to fill these knowledge gaps. These focused mechanistic studies will be complemented with phosphoproteomic and genetic screens designed to better understand the signaling landscape during mitosis. Although some wiring aspects of the signaling networks may be found to vary between organisms, these studies will have a major impact for understanding how cytokinesis can in principle be entrained with other events of mitosis to protect genome integrity.